The Chemokine Fractalkine Inhibits Fas-Mediated Cell Death
of Brain Microglia
Stefen A. Boehme,1Francisco M. Lio, Dominique Maciejewski-Lenoir, Kevin B. Bacon,2and
Paul J. Conlon
Fractalkine is a CX3C-family chemokine, highly and constitutively expressed on the neuronal cell surface, for which a clear CNS
physiological function has yet to be determined. Its cognate receptor, CX3CR-1, is constitutively expressed on microglia, the
brain-resident macrophages; however, these cells do not express fractalkine. We now show that treatment of microglia with
fractalkine maintains cell survival and inhibits Fas ligand-induced cell death in vitro. Biochemical characterization indicates that
this occurs via mechanisms that may include 1) activation of the phosphatidylinositol-3 kinase/protein kinase B pathway, resulting
in phosphorylation and blockade of the proapoptotic functions of BAD; 2) up-regulation of the antiapoptotic protein Bcl-xL; and
3) inhibition of the cleavage of BH3-interacting domain death agonist (BID). The observation that fractalkine serves as a survival
factor for primary microglia in part by modulating the protein levels and the phosphorylation status of Bcl-2 family proteins
reveals a novel physiological role for chemokines. These results, therefore, suggest that the interaction between fractalkine and
CX3CR-1 may play an important role in promoting and preserving microglial cell survival in the CNS. The Journal of Immu-
nology, 2000, 165: 397–403.
erning the trafficking of leukocytes, as well as in modulating cell
adhesion, T lymphocyte activation, phagocytosis, cytokine secre-
tion, angiogenesis, viral pathogenesis, and proliferation. At present
there are four subfamilies of chemokines, distinguished on the rel-
ative position of highly conserved cysteine residues in the amino
terminus of the peptide. Fractalkine is the only chemokine identi-
fied to date that possesses a CX3C motif. Fractalkine is also unique
in that the chemokine domain can be tethered to the cell surface via
a mucin stalk attached to a transmembrane and intracellular do-
main (3, 4). Thus, fractalkine is biologically active as either a
95-kDa membrane-anchored protein, or a secreted chemokine
upon protease cleavage from the mucin stalk. The tissue expres-
sion pattern of fractalkine is also unique, as it is also expressed in
tissues of nonhemopoietic origin such as kidney, lung, and heart.
Furthermore, it is highly and constitutively expressed in the CNS
by neurons, and is up-regulated by injurious stimuli in neurons, or
by TNF-? or IL-1? in astrocytes (5, 6). The fractalkine receptor,
CX3CR-1, is highly expressed by microglia (5, 7–9). It has been
shown to induce cell migration in primary microglial cells, as well
as activate the p42/p44 mitogen-activated protein kinase and phos-
phatidylinositol-3 (PI-3)3kinase/protein kinase B (PKB) signal
transduction pathways (6).
hemokines compose a superfamily of chemoattractant
proteins that generally range from 7 to 14 kDa (1, 2).
They have been shown to play an important role in gov-
Microglia have been shown to express both Fas (CD95) and Fas
ligand (CD95L), and Fas-mediated apoptosis has been implicated
in the pathogenesis of various CNS diseases, such as ischemia-
reperfusion injury (10, 11), multiple sclerosis (12–14), and its ro-
dent counterpart, experimental autoimmune encephalomyelitis
(15–17). However, the exact role of Fas/Fas ligand in the pathol-
ogy of multiple sclerosis/experimental autoimmune encephalomy-
elitis is unclear. Thus, as the Fas/Fas ligand interaction plays a role
in maintaining hemopoietic homeostasis, it may also contribute to
the pathogenesis of various disease processes (18). The binding of
Fas ligand to the cell surface receptor Fas initiates a cascade of
events leading to activation of various caspases in conjunction
with modulation of Bcl-2 family proteins (19). The latter results in
compromised mitochondrial function and integrity, which may
contribute directly to the apoptotic process or be a result of caspase
8 and caspase 3 activation (19, 20). Specifically, the proapoptotic
Bcl-2 family member BAD has the ability to complex with either
Bcl-2 or Bcl-xLand antagonize their antiapoptotic function (21).
This process is regulated in part by PKB/Akt, which can phos-
phorylate BAD at serine 136, rendering this proapoptotic protein
inactive (22–25). Additionally, PKB/Akt can act via NF-?B to
block apoptosis (26, 27). The proapoptotic protein BID is cleaved
upon Fas ligand binding, allowing heterodimerization with Bcl-2
or Bcl-xL, and provides another pathway resulting in the abroga-
tion of Bcl-2 or Bcl-xLantiapoptotic function (28).
The expression of fractalkine by neurons, and CX3CR-1 by mi-
croglial cells suggests that a paracrine interaction exists; however,
no definitive CNS function has yet been elucidated. We have re-
cently shown that fractalkine activates the PKB/Akt signaling
pathway in primary microglia (6). As PKB/Akt has been shown to
be a survival factor in a number of systems, we asked whether
fractalkine stimulation could promote survival of primary brain
microglia, and furthermore block Fas-mediated microglial cell
death. We show in this work for the first time that the chemokine
fractalkine can increase microglial cell survival and block
Fas-induced programmed cell death of primary cells. This novel
function provides crucial insight into the role fractalkine plays in
Neurocrine Biosciences, Inc., San Diego, CA 92121
Received for publication January 24, 2000. Accepted for publication April 12, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Address correspondence and reprint requests to Dr. Stefen A. Boehme, Neurocrine
Biosciences, Inc., 10555 Science Center Drive, San Diego, CA 92121-1102. E-mail
2Current address: Bayer Yakuhin, Ltd., Research Center Kyoto, Soraku-gun, Kyoto
3Abbreviations used in this paper: PI-3, phosphatidylinositol-3; BID, BH3-interact-
ing domain death agonist; DAPI, 4?,6?-diamidino-2-phenylindole; PKB, protein ki-
Copyright © 2000 by The American Association of Immunologists0022-1767/00/$02.00
regulating CNS homeostasis, as well as demonstrating evidence of
Materials and Methods
All reagents were obtained from Sigma (St. Louis, MO), unless otherwise
specified. Animal experimentation was approved by the Institutional Ani-
mal Care and Use Committee (IACUC) before implementation.
Primary microglial cell cultures
Microglial cell cultures were established as previously described (6).
Briefly, cortexes from newborn Sprague Dawley rats (Charles River, Bos-
ton, MA) were isolated, mechanically dissociated, and plated at a density
of one brain/T75 flask (Costar, Charlotte, NC) in DMEM (Mediatech, Tus-
tin, CA) containing 10% FCS (HyClone, Logan, UT). Once confluent, the
cells were left for 5–7 days without changing the media to favor microglia
proliferation. The mixed glial cells were then shaken for 6–20 h at 225
rpm. The supernatant, containing an enriched population of microglia, was
pelleted and the cells were replated in DMEM ? 10% FCS. After 2 h, the
cells were manually shaken and the medium was replaced with DMEM ?
10% FCS containing 200 U/ml of both GM-CSF (R&D Systems, Minne-
apolis, MN) and M-CSF (R&D Systems). The adherent cells (?95% pure
microglia) were grown for an additional 48 h before assaying. At this point,
they were detached with Versene (Life Technologies, Gaithersburg, MD),
replated in DMEM ? 10% FCS, and cultured in experiments, as described.
It should be noted that no exogenous growth factors were added during the
experimental procedures, except where indicated (Fig. 1).
Cell death detection ELISA
A total of 5 ? 104microglia/well was seeded into a 96-well flat-bottom
plate (Costar) following the described conditions, and each condition was
done in triplicate. Before the addition of cells, tissue culture plate wells
were coated by the addition of the indicated concentration of fractalkine
(chemokine portion only; R&D Systems) diluted in PBS (Mediatech), and
incubated at 4°C overnight. Control wells received PBS. Following incu-
bation, the wells were washed once with PBS; the cells were then added
and cultured for the indicated time periods. Soluble Fas ligand was ob-
tained from Upstate Biotechnology (Lake Placid, NY). Eighteen hours
postplating, the cells were harvested, and the oligosomal DNA was quan-
titated using the Cell Death Detection ELISA (Roche Diagnostics, India-
napolis, IN), according to the manufacturer’s protocol.
Microglial cells were cultured in 24-well plates at 5 ? 105cells/well for
18 h under the various conditions. They were then isolated using Versene,
pelleted onto slides, and fixed using 4% paraformaldehyde, followed by a
?20°C methanol wash. The cells were then stained with DAPI (Calbio-
chem, La Jolla, CA) at a 1 ?g/ml concentration for 15 min, and rinsed in
PBS. Photomicrographs were taken using a Nikon Eclipse TE300
Western blot analysis
Microglia were seeded into 24-well plates at a concentration of 5 ? 105/
well at the described conditions. Following an 18-h incubation, the micro-
glia were harvested with Versene and washed twice with PBS. The cells
were subsequently suspended in lysis solution (1% Nonidet P-40, 50 mM
Tris-HCl (pH 8), 150 mM NaCl, 0.25% deoxycholate, and 5 mM EDTA)
containing protease and phosphatase inhibitors (10 ?g/ml aprotinin, 10
?g/ml leupeptin, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM EGTA,
100 ?M ?-glycerophosphate, 10 mM sodium flouride, and 1 mM tetraso-
dium phosphate). Cells were incubated on ice for 30 min and centrifuged
to pellet insoluble material, and the protein was quantified using the bicin-
choninic acid assay (Pierce, Rockford, IL). Western blot analysis was con-
ducted as previously described, using Tris-glycine acrylamide gels (Novex,
San Diego, CA) (29). Hybridized proteins were visualized using enhanced
chemiluminescence reagents (Pierce) and Biomax MR autoradiography
film (Eastman Kodak, Rochester, NY). The following primary Abs were
used to probe the Western blots: anti-PKB? polyclonal Ab (Upstate Bio-
technology), anti-BAD Ab (New England Biolabs, Beverly, MA), anti-
phosphoserine Ab (Zymed Laboratories, South San Francisco, CA), anti-
Bcl-xL(Transduction Laboratories, Lexington, KY), anti-Bax Ab (Upstate
Biotechnology), and anti-BID polyclonal Ab (Santa Cruz Biotechnology,
Santa Cruz, CA). Equal amounts of protein (25 ?g) were loaded into
Microglial cell lysates were collected as described above and used to carry
out the pulldown experiments. One microgram of Bcl-2-GST fusion pro-
tein coupled to agarose beads (Upstate Biotechnology), or 1 ?g anti-BAD
Abs and protein G-coated agarose beads (Santa Cruz Biotechnology) were
added to 5 ?g of fresh cell lysates, and the immunoprecipitation was con-
ducted overnight at 4°C on an orbital rocker. The agarose beads were
subsequently pelleted by centrifugation, washed twice with lysis buffer
containing the protease and phosphatase inhibitors, and analyzed by West-
Measurement of Akt/PKB activity
Assay for Akt/PKB activity was performed as previously described (6).
Microglial cells were stimulated with fractalkine and the PI-3 kinase in-
hibitor LY294002 (Calbiochem) at the indicated concentrations. PKB was
immunoprecipitated using an anti-rat PKB? polyclonal Ab (Santa Cruz
Biotechnology) and precipitated with protein G agarose beads (Santa Cruz
Biotechnology). Kinase reactions were performed in kinase reaction buffer
(20 mM HEPES (pH 7.4), 10 mM MgCl2, 10 mM MnCl2, 0.05 mg/ml
histone 2B, 5 ?M ATP, 1 mM DTT, and 10 ?Ci [?-32P]ATP) for 30 min
at 30°C. The reactions were halted by the addition of an equal volume of
2? Laemmli sample buffer and boiling. Histone 2B phosphorylation was
resolved using a 16% Tris glycine gel (Novex) and visualized by autora-
diography. Equal loading of Akt/PKB was detected by Western analysis, as
Cell viability and proliferation
Microglia were cultured in 96-well plates (5 ? 104cells/well) and were
either untreated, seeded onto wells coated with 100 nM fractalkine, or
cultured in the presence of 200 U/ml M-CSF and GM-CSF (R&D Sys-
tems). Mitochondrial function was used as a measure of cell survival, and
after 44 h of culture, an MTT assay was performed, according to the man-
ufacturer’s instructions (Roche Diagnostics). After 24 h, parallel cultures
were pulsed with 1 ?Ci of [3H]TdR (6.7 mCi/mmol; New England Nu-
clear, Boston, MA), and [3H]TdR incorporation was measured by betaplate
scintigraphy at 48 h. Each experimental point was done in quadruplicate,
and five independent experiments were performed. Dr. Lili Feng (Scripps
Research Institute, La Jolla, CA) kindly provided the neutralizing
Two-tailed Student’s t test was used to determine whether differences ob-
served were significant. A p value ?0.05 was considered significant.
Fractalkine acts as a survival factor for primary brain
In analyses of fractalkine effects on primary rat microglial cells in
vitro, we observed that cells grown in the presence of immobilized
fractalkine (as described in Materials and Methods) showed in-
creased viability over time when compared with unstimulated cells
by MTT assay (Fig. 1). Both microglial cell survival and mito-
chondrial function were enhanced in the presence of fractalkine,
and a specific blocking Ab to the receptor, CX3CR-1, abrogated
this effect. No comparable effects were seen with 20 other chemo-
kines tested (data not shown). In contrast to microglia treated with
both M-CSF/GM-CSF, fractalkine-treated cells proliferated mini-
mally (Fig. 1; [3H]thymidine). These data suggest that fractalkine
serves a unique function compared with other chemokines, that of
a survival factor for microglial cells in vitro.
Fractalkine can block Fas-mediated apoptosis of microglia
To further analyze the survival-promoting effects of fractalkine,
we asked whether fractalkine could antagonize Fas-induced pro-
grammed cell death. Microglia were stimulated with plate-bound
fractalkine (immobilized chemokine domain alone) and various
398FRACTALKINE INHIBITS Fas-MEDIATED DEATH OF MICROGLIA
doses of soluble Fas ligand. After an 18-h incubation period (de-
termined through time-course experiments), cell viability was as-
sessed. Microscopic examination of the microglia revealed exten-
sive cell death in samples treated only with Fas ligand, as
evidenced by cell aggregation and condensed cytoplasm (data not
shown). DAPI staining of the cells showed that Fas ligand-treated
microglia had undergone chromatin condensation and nuclear
fragmentation (Fig. 2A). In contrast, untreated, fractalkine-treated
or fractalkine and Fas ligand-treated microglia had substantially
fewer cells displaying condensed chromatin. In parallel, microglial
cells that did not receive fractalkine treatment had an approximate
4–5-fold increase in the amount of oligosomal DNA, which is
indicative of apoptosis, and this effect was dependent on the dose
of Fas ligand added (Fig. 2B). A dose titration of fractalkine re-
vealed that 10 nM was sufficient to effectively inhibit Fas-mediated
apoptosis (Fig. 2C). Additional evidence that this effect was unique
to fractalkine was obtained using eotaxin. Functional CCR-3 re-
ceptors are widely expressed on microglia (30), and both fracta-
lkine and eotaxin stimulate phosphorylation of extracellular signal-
related kinase 1 and 2, demonstrating an overlap in signal
transduction pathways (Ref. 6; and S. A. Boehme and K. B. Bacon,
unpublished observations). Unlike fractalkine, eotaxin was unable
to rescue Fas-induced cell death (Fig. 2B).
DNA content analysis of microglia stimulated with fractalkine,
Fas ligand, or both revealed a 5-fold increase in the amount of cells
with hypodiploid DNA in Fas ligand-treated samples compared
with fractalkine/Fas ligand-treated microglia (Table I). A total of
4.2% of fractalkine and fas ligand-treated cells had a hypodiploid
DNA content contrasted with 20.6% of the Fas ligand-treated cells.
These data are consistent with our results measuring oligosomal
DNA, and are not due to fractalkine-induced down-regulation of
Fas from the microglial cell surface (data not shown). Addition-
ally, this analysis further demonstrated fractalkine’s survival factor
effect on microglia, as 7.21% of untreated microglia had hypodip-
loid DNA content compared with just 1.93% of the fractalkine-
treated cells (Table I). DNA content analysis also suggested that
fractalkine may be inducing a G1cell cycle block in microglia, as
a substantially greater number of cells were in the G1phase of the
cell cycle following 18 h of fractalkine stimulation compared with
controls (Table I). Collectively, these results demonstrated that
under the conditions tested, fractalkine stimulation of microglia
had a pronounced survival effect.
Stimulation of microglia with fractalkine leads to PKB/Akt
activation and phosphorylation of BAD
The significance of these effects on cell survival correlates with in
vitro biochemical signaling. Previously, we demonstrated that
fractalkine activates the PI-3 kinase/PKB pathway in primary mi-
croglia (6), and this might account for fractalkine acting as a sur-
vival factor. Therefore, we tested whether blockade of this path-
way by the PI-3 kinase inhibitor LY294002 could reverse the
protective effect of fractalkine (31). The addition of 1 ?M
LY294002 to microglial cell cultures effectively blocked the ki-
nase activity of PKB (Fig. 3A); however, it only partially antago-
nized the protective effect of fractalkine (Fig. 3B). Similar effects
were observed using another PI-3 kinase antagonist, wortmannin,
at several concentrations (data not shown). This observation illus-
trates that activation of the PI-3 kinase/PKB pathway plays a role
in fractalkine’s ability to block Fas-mediated apoptosis, although
only partial protection was seen under various doses, suggesting
multiple pathways mediate fractalkine’s protective effect.
To further biochemically characterize the role of the PI-3 ki-
nase/PKB pathway, microglia were treated for 18 h with fractal-
kine and Fas ligand, separately or in combination, BAD protein
was immunoprecipitated, and phosphoserine levels were examined
by Western blot analysis (Fig. 4A). This revealed that microglial
cells treated with fractalkine had substantially higher levels of
phosphoserine-BAD protein, indicative of inhibition of its pro-
apoptotic function. When the immunoblot was reprobed, compa-
rable levels of BAD protein were found under all conditions tested
(Fig. 4B). Additionally, we tested whether BAD could het-
erodimerize with Bcl-2 under these conditions; Western blots of
pulldown experiments using a Bcl-2-GST fusion protein were
probed for BAD. The levels of BAD protein precipitated with the
Bcl-2 fusion protein were reduced in untreated, fractalkine-treated,
or fractalkine and Fas ligand-treated microglia, compared with Fas
ligand-stimulated microglia, further suggesting that BAD had been
phosphorylated and lost its ability to heterodimerize (Fig. 4C).
Fractalkine stimulation can modulate expression of Bcl-2 family
As the regulatory pathway of BAD phosphorylation did not appear
to be the sole regulator of apoptosis (Fig. 3B), the role of other
Bcl-2 family proteins was explored by measuring levels of the
croglial survival. MTT assay (?) and
[3H]thymidine incorporation (f) measur-
ing cell viability and proliferation, respec-
tively. Microglia were either untreated
(control) or treated for 48 h with 100 nM
fractalkine (chemokine domain), or 200
U/ml M-CSF and GM-CSF in the pres-
ence or absence of neutralizing concentra-
the last 4 h of the experiment to measure
mitochondrial metabolic function as an in-
dicator of cell survival. Cells were pulsed
after 24 h, and [3H]thymidine incorpora-
tion was measured at 48 h to assess pro-
liferation. All results have been normal-
ized to control levels (100%) ? SEM, and
a representative of five separate experi-
ments is shown. Statistical differences
were significant between the various sam-
ples; ?, denotes p ? 0.001, and ??, indi-
cates p ? 0.005.
Fractalkine promotes mi-
399The Journal of Immunology
antiapoptotic protein Bcl-xLand the proapoptotic protein Bax. The
intracellular ratio of these proteins is thought to dictate cell fate
(32). Our results show that Bcl-xLis up-regulated in microglia
treated with fractalkine for 18 h; conversely, Bax protein levels are
reduced (Fig. 5, A and B). Additionally, levels of the proapoptotic
protein BID were examined (28). BID has been shown to be a
critical mediator in the mitochondrial amplification loop, resulting
in Fas-induced apoptosis (33). We observed BID to be cleaved and
treated, treated with 100 ng/ml soluble Fas ligand, 10 nM immobilized fractalkine (chemokine domain), or both for 18 h. Arrows indicate cells with clear
morphological nuclear changes associated with apoptosis, as the apoptotic nuclei have condensed chromatin and nuclear blebbing. b, Oligosomal DNA
content was measured in microglia challenged with various doses of soluble Fas ligand receiving either no treatment (?), 10 nM plate-bound fractalkine
(chemokine domain; Œ), or soluble 10 nM eotaxin (ƒ) for 18 h. The results shown are the mean of four independent experiments ? SEM. c, Fractalkine
protection of Fas ligand-treated microglia is dose dependent. Microglia were treated as above with various doses of plate-bound fractalkine, and oligosomal
DNA content was determined after 18 h. Representative results from three independent experiments are shown ? SEM.
Fractalkine blocks Fas-induced programmed cell death. a, Photomicrographs (?1000) of DAPI-stained microglial nuclei that were un-
400 FRACTALKINE INHIBITS Fas-MEDIATED DEATH OF MICROGLIA
stimulated microglia and plays a role in fractalkine inhibition of Fas-me-
diated microglial cell death. A, PKB immunoprecipitates from microglia
treated with either PBS or 10 nM plate-bound fractalkine with various
concentrations of the PI-3 kinase inhibitor LY294002 were used in in vitro
kinase reactions using histone 2B as an exogenous substrate for PKB. To
control for equal amounts of Akt/PKB immunoprecipitated, the immuno-
blot was probed with anti-Akt/PKB? polyclonal Abs (lower panel). B,
Quantitation of oligosomal DNA detected from either untreated microglial
cells or microglia stimulated in the presence or absence of 1 ?M
LY294002 and 10 nM immobilized fractalkine. All conditions received 50
ng/ml of soluble Fas ligand for 18 h. Results shown are the mean from
three separate experiments ? SEM.
The PI-3 kinase/PKB pathway is activated in fractalkine-
stimulation in microglia. Western blot analysis of BAD expression and
posttranslational phosphorylation in microglia treated for 18 h with either
10 nM plate-bound fractalkine (chemokine domain), 100 ng/ml soluble Fas
ligand, or in combination. A, BAD protein was immunoprecipitated from
fresh lysates, and the immunoblot was probed with anti-phosphoserine Ab
(n ? 3; a representative Western blot is shown for A, B, and C). B, The
same immunoblot was subsequently reprobed with anti-BAD Abs as a
control to demonstrate similar levels of BAD protein were immunopre-
cipitated in each sample (n ? 3). This approach was taken because the
cross-reactivity of the anti-phospho-BAD Abs tested was suboptimal
against rat cell lysates. C, Western blot analysis of BAD protein levels
using a Bcl-2-GST fusion protein in a pulldown experiment. Microglia
were treated as above, and the fresh cell lysates were incubated with 1 ?g
of a Bcl-2-GST fusion protein linked to agarose beads and precipitated.
Western blots were subsequently probed with anti-BAD Abs, indicating the
relative amounts of BAD protein heterodimerized with Bcl-2 (n ? 3).
Histograms of the densitometry readings are shown for each blot, and are
normalized to untreated samples.
BAD protein is serine phosphorylated upon fractalkine
Table I. Fractalkine blocks Fas-mediated programmed cell death-induced DNA degradation in primary
Percentage of Cells in:
7.21 ? 0.15
1.93 ? 0.23
20.62 ? 0.23
4.24 ? 0.20
61.14 ? 0.65
80.85 ? 1.68
58.66 ? 6.39
71.66 ? 5.35
25.1 ? 6.39
11.69 ? 1.45
15.86 ? 0.45
19.27 ? 5.28
8.64 ? 0.45
6.32 ? 2.46
6.01 ? 0.27
6.41 ? 0.18
aMicroglial cells were either untreated, or stimulated with 10 nM fractalkine, 100 ng/ml soluble Fas ligand, or both for 18 h,
and harvested. The DNA content was determined by staining ethanol-fixed cells with propidium iodide and flow cytometric
analysis. The ?G1cells contain hypodiploid DNA content indicative of apoptosis. The values obtained are from three exper-
iments (?SEM). Differences between the ?G1fraction of the various samples were significant (p ? 0.005).
401The Journal of Immunology
activated in Fas ligand-treated cultures of microglia; however, the
amount of activated protein was significantly reduced in cells stim-
ulated with fractalkine (Fig. 5C). These results illustrate that frac-
talkine inhibition of Fas-induced microglial cell death is mediated
at least in part through specific antiapoptotic signaling. Taken to-
gether with the survival effect measured by mitochondrial function
(MTT assay, Fig. 1), these observations suggest that one mecha-
nism by which fractalkine is acting to suppress Fas-mediated
apoptosis may be by maintaining mitochondrial integrity.
The ability of chemokines to elucidate various cellular responses,
beyond the induction of chemotaxis, is becoming more defined and
broadened (1). In this work, we explored the function of fractal-
kine in the CNS, as this unique chemokine is highly and consti-
tutively expressed by neurons. Conversely, the receptor for frac-
talkine, CX3CR-1, is expressed at high levels by microglial cells
(30,000–50,000 sites/microglial cell compared with less than
5,000 sites/peripheral blood leukocyte; K. B. Bacon, unpublished
observation). Our data demonstrate for the first time that fracta-
lkine can act as a survival factor for primary microglial cells. Thus,
fractalkine may provide the physiological cue necessary for the
stability of the microglial cell population within the CNS (34). The
results further show that fractalkine exerts these effects in part by
1) activating the PI-3 kinase/PKB pathway, and 2) directly mod-
ulating both levels and activation states of Bcl-2 family member
proteins. Therefore, one pathway by which fractalkine may act as
a survival factor is by preserving mitochondrial integrity and func-
tion in microglial cells. Experiments are ongoing, examining the
effect of fractalkine stimulation in the absence or presence of Fas
ligand treatment on caspase activation. Additionally, DNA content
analysis of microglial cells suggests that fractalkine appears to
induce a G1cell cycle block. It has been demonstrated that TCR-
mediated apoptosis of activated CD4?T lymphocytes occurs pre-
dominantly via a Fas ligand/Fas interaction, and blocking the cells
in the G1phase of the cell cycle can abrogate this effect (35–37).
This observation raises the question of whether fractalkine may
also be acting via the cell cycle to inhibit Fas-mediated cell death,
and this hypothesis is currently being tested.
The Fas ligand/Fas pathway has been shown to be involved in
various CNS diseases (10–17). Normally, Fas is only weakly ex-
pressed in the brain, but is up-regulated in brain tissues from
stroke, multiple sclerosis, and Alzheimer’s patients, suggesting it
may play a role in various CNS pathological states (10, 38). In
these diseases, there is a concomitant release of Fas ligand into the
CNS environment by various cell types, including microglia (10,
14). We show in this work that fractalkine can inhibit Fas-medi-
ated death of primary microglial cells in vitro. Although fractal-
kine is constitutively expressed in the brain, deleterious stimuli
further induce fractalkine expression by neurons (5). Additionally,
the proinflammatory cytokines TNF-? and IL-1? trigger fractal-
kine expression by astrocytes (6). Taken together, these observa-
tions suggest a critical role of fractalkine in various CNS disease
states, and one function may be to promote microglial cell
Microglia are brain-resident macrophage, and they perform a
critical role as phagocytic cells. Furthermore, their anatomic loca-
tion close to the blood brain barrier, and their ability to secrete
inflammatory cytokines, reactive oxygen intermediates, NO, and
Fas ligand allow microglia to serve as critical regulators of CNS
inflammation (34). Thus, microglia play a key role mediating CNS
tissue damage, as this cell type has the ability to both amplify and
control CNS pathology, both in the early stages of inflammation
and the later phase of tissue repair.
In light of microglial function in the CNS both in homeostasis
and disease states, our results demonstrate a novel and potentially
critical function of fractalkine. These results illustrate a physio-
logically relevant role for this constitutively expressed chemokine
in the brain. In summary, the data presented demonstrate a novel
biological precedent for a chemokine, and provide insight into ba-
sic cellular interactions between neurons and microglia in both
normal and pathological conditions within the CNS. These obser-
vations may, in turn, open up new avenues for therapeutic gain in
reducing CNS damage in various diseases by modulating the frac-
We thank Drs. Guy Salvesen and Nick Ling for insightful discussions, and
Drs. David Alleva, Paul Crowe, Kenny MacKay, Greg Naeve, Robert
Petrowski, and David Schwartz for critical reading of the manuscript. We
further thank Dr. Greg Naeve for technical help and Joelle Eggold for
expert graphics assistance.
1. Baggiolini, M., B. Dewald, and B. Moser. 1997. Human chemokines: an update.
Annu. Rev. Immunol. 15:675.
2. Rollins, B. J. 1997. Chemokines. Blood 90:909.
and down-regulates proapoptotic Bax and activated BID. Microglia were
stimulated with 10 nM immobilized fractalkine (chemokine domain), 100
ng/ml Fas ligand, or both for 18 h. Cell lysates were examined by Western
analysis for protein expression of A, Bcl-xL(n ? 4); B, Bax (n ? 4); and
C, activated BID (n ? 3). The Ab used to probe for BID was specific for
the p15-activated form. Densitometric analysis of the Western blot is
Fractalkine up-regulates the antiapoptotic protein Bcl-xL
402 FRACTALKINE INHIBITS Fas-MEDIATED DEATH OF MICROGLIA
3. Bazan, J. F., K. B. Bacon, G. Hardiman, W. Wang, K. Soo, D. Rossi,
D. R. Greaves, A. Zlotnick, and T. J. Schall. 1997. A new class of membrane-
bound chemokine with a CX3C motif. Nature 385:640.
4. Pan, Y., C. Lloyd, H. Zhou, S. Dolich, J. Deeds, J. A. Gonzalo, J. Vath,
M. Gosselin, J. Ma, B. Dussault, et al. 1997. Neurotactin, a membrane-anchored
chemokine up-regulated in brain inflammation. Nature 387:611.
5. Harrison, J. K., Y. Jiang, S. Chen, Y. Xia, D. Maciejewski, R. K. McNamara,
W. J. Streit, M. N. Salafranca, S. Adhikari, D. A. Thompson, et al. 1998. Role for
neuronally derived fractalkine in mediating interactions between neurons and
CX3CR-1-expressing microglia. Proc. Natl. Acad. Sci. USA 95:10896.
6. Maciejewski-Lenoir, D., S. Chen, L. Feng, R. Maki, and K. B. Bacon. 1999.
Characterization of fractalkine in rat brain cells: migratory and activation signals
for CX3CR-1-expressing microglia. J. Immunol. 163:1628.
7. Imai, T., K. Hieshima, C. Haskell, M. Baba, M. Nagira, M. Kakizaki, S. Takagi,
H. Nomiyama, T. J. Schall, and O. Yoshie. 1997. Identification and molecular
characterization of fractalkine receptor CX3CR-1, which mediates both leukocyte
migration and adhesion. Cell 91:521.
8. Harrison, J. K., C. M. Barber, and K. R. Lynch. 1994. cDNA cloning of a G-
protein-coupled receptor expressed in rat spinal cord and brain related to che-
mokine receptors. Neurosci. Lett. 169:85.
9. Combadiere, C., S. K. Ahuja, and P. M. Murphy. 1995. Cloning, chromosomal
localization, and RNA expression of a human ? chemokine receptor-like gene.
DNA Cell Biol. 14:673.
10. Vogt, M., M. K. A. Bauer, D. Ferrari, and K. Schulze-Osthoff. 1998. Oxidative
stress and hypoxia/reoxygenation trigger CD95 ligand expression in microglial
cells. FEBS Lett. 429:67.
11. Matsuyama, T., R. Hata, M. Tagaya, Y. Yamamoto, T. Nakajima, J. Furuyama,
A. Wanaka, and M. Sugita. 1994. Fas antigen mRNA induction in postischemic
murine brain. Brain Res. 657:342.
12. Dowling, P., G. Shang, S. Raval, J. Menonna, S. Cook, and W. Husar. 1996.
Involvement of the CD95 receptor/ligand system in multiple sclerosis brain.
J. Exp. Med. 184:1513.
13. Bonetti, B., and C. S. Raine. 1997. Multiple sclerosis: oligodendrocytes display
cell-death-related molecules in situ but do not undergo apoptosis. Ann. Neurol.
14. D’Souza, S. D., B. Bonetti, V. Balasingam, N. R. Cashman, P. A. Barker,
A. B. Troutt, C. S. Raine and J. P. Antel. 1996. Multiple sclerosis: Fas signaling
in oligodendrocyte cell death. J. Exp. Med. 184:2361.
15. Bonetti, B., J. Pohl, Y. L. Gao, and C. S. Raine. 1997. Cell death during auto-
immune demyelination: effector but not target cells are eliminated by apoptosis.
J. Immunol. 159:5733.
16. Sabelko, K. A., K. A. Kelly, M. H. Nahm, A. H. Cross, and J. H. Russell. 1997.
Fas and Fas ligand enhance the pathogenesis of experimental allergic encepha-
lomyelitis, but are not essential for immune privilege in the central nervous sys-
tem. J. Immunol. 159:3096.
17. Waldner, H., R. A. Sobel, E. Howard, and V. K. Kuchroo. 1997. Fas- and FasL-
deficient mice are resistant to induction of autoimmune encephalomyelitis. J. Im-
18. Lenardo, M. J. 1996. Fas and the art of lymphocyte maintenance. J. Exp. Med.
19. Peter, M. E., and P. H. Krammer. 1998. Mechanisms of CD95-mediated apopto-
sis. Curr. Opin. Immunol. 10:545.
20. Lenardo, M. J., F. K.-M. Chan, F. Hornung, H. McFarland, R. Siegel, J. Wang,
and L. Zheng. 1999. Mature T lymphocyte apoptosis-immune regulation in a
dynamic and unpredictable antigenic environment. Annu. Rev. Immunol. 17:221.
21. Yang, E., J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer.
1995. BAD, a heterodimeric partner for Bcl-xLand Bcl-2, displaces Bax and
promotes cell death. Cell 80:285.
22. Kauffmann-Zeh, A., P. Rodriguez-Viciana, E. Ulrich, C. Gilbert, P. Coffer,
J. Downward, and G. Evan. 1997. Suppression of c-Myc-induced apoptosis by
Ras signalling through PI(3)K and PKB. Nature 385:544.
23. Datta, S. R., H. R. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, and
M. E. Greenberg. 1997. Akt phosphorylation of BAD couples survival signals to
the cell-intrinsic death machinery. Cell 91:231.
24. Zha, J., H. Harada, E. Yang, J. Jockel, and S. J. Korsmeyer. 1996. Serine phos-
phorylation of death agonist BAD in response to survival factor results in binding
to 14-3-3 not Bcl-xL. Cell 87:619.
25. Del Peso, L., M. Gonza ´lez-Garcı ´a, C. Page, R. Herrera, and G. Nun ˜ez. 1997.
Interleukin-3-induced phosphorylation of BAD through protein kinase Akt. Sci-
26. Ozes, O. N., L. D. Mayo, J. A. Gustin, S. R. Pfeffer, L. M. Pfeffer, and
D. B. Donner. 1999. NF-?B activation by tumor necrosis factor requires the AKT
serine-threonine kinase. Nature 401:82.
27. Romashkova, J. A., and S. S. Makarov. 1999. NF-?B is a target of AKT in
anti-apoptotic PDGF signalling. Nature 401:86.
28. Luo, X., I. Budihardo, H. Zou, C. Slaughter, and X. Wang. 1998. Bid, a Bcl-2
interacting protein, mediates cytochrome c release from mitochondria in response
to activation of cell surface death receptors. Cell 94:481.
29. Boehme, S. A., S. K. Sullivan, P. D. Crowe, M. Santos, P. J. Conlon,
P. Sriramarao, and K. B. Bacon. 1999. Activation of mitogen-activated protein
kinase regulates eotaxin-induced eosinophil migration. J. Immunol. 163:1611.
30. He, J., Y. Chen, M. Farzan, H. Choe, A. Ohagen, S. Gartner, J. Busciglio,
X. Yang, W. Hofmann, W. Newman, et al. 1997. CCR3 and CCR5 are co-
receptors for HIV-1 infection of microglia. Nature 385:645.
31. Vlahos, C. J., W. F. Matter, K. Y. Hui, and R. F. Brown. 1994. A specific
inhibitor of phosphatidylinositol 3-kinase LY294002. J. Biol. Chem. 269:5241.
32. Oltvai, Z. N., and S. J. Korsmeyer. 1994. Checkpoints of dueling dimers foil
death wishes. Cell 79:189.
33. Yin, X.-M., K. Wang, A. Gross, Y. Zhao, S. Zinkel, B. Klocke, K. A. Roth, and
S. J. Korsmeyer. 1999. Bid-deficient mice are resistant to Fas-induced hepato-
cellular apoptosis. Nature 400:886.
34. Gehrmann, J., Y. Matsumoto, and G. W. Kreutzberg. 1995. Microglia: intrinsic
immuneffector cell of the brain. Brain Res. Rev. 20:269.
35. Zheng, L., G. Fisher, R. E. Miller, J. Peschon, D. H. Lynch, and M. J. Lenardo.
1995. Induction of apoptosis in mature T cells by tumor necrosis factor. Nature
36. Boehme, S. A., and M. J. Lenardo. 1993. Propriocidal apoptosis of mature T
lymphocytes occurs at S phase of the cell cycle. Eur. J. Immunol. 23:1552.
37. Boehme, S. A., and M. J. Lenardo. 1993. Ligand-induced apoptosis of mature T
lymphocytes occurs at distinct stages of the cell cycle. Leukemia 7:545.
38. Nishimura, T., H. Akiyama, S. Yonehara, H. Kondo, K. Ikeda, M. Kato, E. Iseki,
and K. Kosaka. 1995. Fas antigen expression in brains of patients with Alzhei-
mer-type dementia. Brain Res. 695:137.
403 The Journal of Immunology